The goal of this proposal is to elucidate the developmental role of the NG2 proteoglycan, a 500 kD cell surface, integral membrane proteoglycan consisting of a 260 kD core polypeptide, N-linked oligosaccharides totaling 40 kD, and chondroitin sulfate chains totaling about 200 kD. During development NG2 is not found on undifferentiated stem cells, but is expressed at a later stage of differentiation on partially committed precursor cells that are still mitotically active. These precursor populations include the O2A glial progenitor cells of the central nervous system, dividing chondroblasts of the developing limb and spinal column, skeletal muscle myoblasts, and dividing capillary epithelial cells. In each case NO2 expression is down-regulated upon terminal differentiation of the precursor cells. These observations suggest that NG2 may be involved in controlling cell proliferation and/or differentiation., Two pathological conditions reinforce this impression. First, reactive astrocytes, which arise in response to neuronal injury, become mitotically active and begin to express NG2. Second, NG2 is up-regulated on malignant glioblastoma cells as well as on the mitotic capillary endothelial cells in the expanding vasculature of these tumors. Our preliminary evidence indicates that NG2 may affect growth control by enhancing responsiveness of cells to growth factors. The proposed research will test the functional role of NG2 in vivo by studying the effects of altered NG2 expression in three lines of transgenic mice. Determinations will be made of the consequences both of abolishing NG2 expression and of over-expressing NG2 in mature cell types that normally down-regulate expression of the proteoglycan. The first objective will be achieved by creating a mutant mouse line in which NG2 is deleted by homologous recombination with a defective NG2 construct. The second objective will be achieved by creating transgenic mice in which the transcription of NG2 is controlled by the regulatory sequences of genes that are expressed specifically in mature cell types derived from NG2-positive progenitors. The O2A glial progenitor system is a good choice for these studies because the tissue-specific activity of the promoters for genes expressed in mature glial cells has been well established. The promoter for myelin basic protein will be used to drive NG2 expression in mature oligodendrocytes and the promoter for the glial fibrillary acidic protein will be used to express NG2 in mature astrocytes. The production, maturation, and functional capabilities of oligodendrocytes and astrocytes will be examined in each of the mouse lines. In the case of the NG2 gene knockout mouse, the development of the other tissues that are normally NG2 positive will also be studied.